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Structural basis of cell wall cleavage by a staphylococcal autolysin.

Zoll S, Pätzold B, Schlag M, Götz F, Kalbacher H, Stehle T - PLoS Pathog. (2010)

Bottom Line: Mutations of key residues in the putative active site result in loss of activity, enabling us to propose a catalytic mechanism.This results in a plausible model of interaction of this ligand not only for AmiE, but also for other PGN-hydrolases that share the same fold.As AmiE active-site mutations also show a severe growth defect, our findings provide an excellent platform for the design of specific inhibitors that target staphylococcal cell separation and can thereby prevent growth of this pathogen.

View Article: PubMed Central - PubMed

Affiliation: Interfaculty Institute for Biochemistry, University of Tübingen, Tübingen, Germany.

ABSTRACT
The major autolysins (Atl) of Staphylococcus epidermidis and S. aureus play an important role in cell separation, and their mutants are also attenuated in virulence. Therefore, autolysins represent a promising target for the development of new types of antibiotics. Here, we report the high-resolution structure of the catalytically active amidase domain AmiE (amidase S. epidermidis) from the major autolysin of S. epidermidis. This is the first protein structure with an amidase-like fold from a bacterium with a gram-positive cell wall architecture. AmiE adopts a globular fold, with several alpha-helices surrounding a central beta-sheet. Sequence comparison reveals a cluster of conserved amino acids that define a putative binding site with a buried zinc ion. Mutations of key residues in the putative active site result in loss of activity, enabling us to propose a catalytic mechanism. We also identified and synthesized muramyltripeptide, the minimal peptidoglycan fragment that can be used as a substrate by the enzyme. Molecular docking and digestion assays with muramyltripeptide derivatives allow us to identify key determinants of ligand binding. This results in a plausible model of interaction of this ligand not only for AmiE, but also for other PGN-hydrolases that share the same fold. As AmiE active-site mutations also show a severe growth defect, our findings provide an excellent platform for the design of specific inhibitors that target staphylococcal cell separation and can thereby prevent growth of this pathogen.

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Results of docking of AmiE with the MTP ligand.(A) Putative interactions between AmiE (grey) and MTP (orange) in the docking model. AmiE residues forming hydrogen bond are colored green, residues making three or more van der Waals interactions are colored blue. Hydrogen bonds are indicated with dashed lines. (B) Surface representation of AmiE with MTP docked into the binding groove. Residues forming hydrogen bonds and van der Waals interactions were calculated using the PISA server (http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html) [39]. The putative locations of glycine residues, which would be attached to the lysine side chain in the natural ligand, are indicated with yellow circles. (C) Schematic representation of interactions between AmiE and MTP. Van der Waals interactions are represented with arcs. The same color scheme for contacting residues was used in panels A, B and C. (D) Summary of the observed hydrogen bonds in the model.
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ppat-1000807-g006: Results of docking of AmiE with the MTP ligand.(A) Putative interactions between AmiE (grey) and MTP (orange) in the docking model. AmiE residues forming hydrogen bond are colored green, residues making three or more van der Waals interactions are colored blue. Hydrogen bonds are indicated with dashed lines. (B) Surface representation of AmiE with MTP docked into the binding groove. Residues forming hydrogen bonds and van der Waals interactions were calculated using the PISA server (http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html) [39]. The putative locations of glycine residues, which would be attached to the lysine side chain in the natural ligand, are indicated with yellow circles. (C) Schematic representation of interactions between AmiE and MTP. Van der Waals interactions are represented with arcs. The same color scheme for contacting residues was used in panels A, B and C. (D) Summary of the observed hydrogen bonds in the model.

Mentions: Efforts to obtain a crystal structure of AmiE with a bound PGN fragment, either through co-crystallization or soaking, were unsuccessful. We therefore undertook molecular docking studies to establish a plausible mode of interaction for the MTP ligand with AmiE (see Methods). The available biochemical and structural data served as constraints for this approach. The best docking solution, which combines the highest docking score with a sensible orientation in the binding groove, is shown in Figure 6. The solution places the carbohydrate moiety in a relatively open, surface-exposed region at one end of the groove, and the tripeptide stem into the groove. Residues in the active site near the zinc ion, which are likely to play a role in catalysis, are positioned near the bond that is cleaved by AmiE. A nucleophilic attack of this bond by an activated water molecule, as shown schematically in Figure 3B, would therefore easily be possible.


Structural basis of cell wall cleavage by a staphylococcal autolysin.

Zoll S, Pätzold B, Schlag M, Götz F, Kalbacher H, Stehle T - PLoS Pathog. (2010)

Results of docking of AmiE with the MTP ligand.(A) Putative interactions between AmiE (grey) and MTP (orange) in the docking model. AmiE residues forming hydrogen bond are colored green, residues making three or more van der Waals interactions are colored blue. Hydrogen bonds are indicated with dashed lines. (B) Surface representation of AmiE with MTP docked into the binding groove. Residues forming hydrogen bonds and van der Waals interactions were calculated using the PISA server (http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html) [39]. The putative locations of glycine residues, which would be attached to the lysine side chain in the natural ligand, are indicated with yellow circles. (C) Schematic representation of interactions between AmiE and MTP. Van der Waals interactions are represented with arcs. The same color scheme for contacting residues was used in panels A, B and C. (D) Summary of the observed hydrogen bonds in the model.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2837410&req=5

ppat-1000807-g006: Results of docking of AmiE with the MTP ligand.(A) Putative interactions between AmiE (grey) and MTP (orange) in the docking model. AmiE residues forming hydrogen bond are colored green, residues making three or more van der Waals interactions are colored blue. Hydrogen bonds are indicated with dashed lines. (B) Surface representation of AmiE with MTP docked into the binding groove. Residues forming hydrogen bonds and van der Waals interactions were calculated using the PISA server (http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html) [39]. The putative locations of glycine residues, which would be attached to the lysine side chain in the natural ligand, are indicated with yellow circles. (C) Schematic representation of interactions between AmiE and MTP. Van der Waals interactions are represented with arcs. The same color scheme for contacting residues was used in panels A, B and C. (D) Summary of the observed hydrogen bonds in the model.
Mentions: Efforts to obtain a crystal structure of AmiE with a bound PGN fragment, either through co-crystallization or soaking, were unsuccessful. We therefore undertook molecular docking studies to establish a plausible mode of interaction for the MTP ligand with AmiE (see Methods). The available biochemical and structural data served as constraints for this approach. The best docking solution, which combines the highest docking score with a sensible orientation in the binding groove, is shown in Figure 6. The solution places the carbohydrate moiety in a relatively open, surface-exposed region at one end of the groove, and the tripeptide stem into the groove. Residues in the active site near the zinc ion, which are likely to play a role in catalysis, are positioned near the bond that is cleaved by AmiE. A nucleophilic attack of this bond by an activated water molecule, as shown schematically in Figure 3B, would therefore easily be possible.

Bottom Line: Mutations of key residues in the putative active site result in loss of activity, enabling us to propose a catalytic mechanism.This results in a plausible model of interaction of this ligand not only for AmiE, but also for other PGN-hydrolases that share the same fold.As AmiE active-site mutations also show a severe growth defect, our findings provide an excellent platform for the design of specific inhibitors that target staphylococcal cell separation and can thereby prevent growth of this pathogen.

View Article: PubMed Central - PubMed

Affiliation: Interfaculty Institute for Biochemistry, University of Tübingen, Tübingen, Germany.

ABSTRACT
The major autolysins (Atl) of Staphylococcus epidermidis and S. aureus play an important role in cell separation, and their mutants are also attenuated in virulence. Therefore, autolysins represent a promising target for the development of new types of antibiotics. Here, we report the high-resolution structure of the catalytically active amidase domain AmiE (amidase S. epidermidis) from the major autolysin of S. epidermidis. This is the first protein structure with an amidase-like fold from a bacterium with a gram-positive cell wall architecture. AmiE adopts a globular fold, with several alpha-helices surrounding a central beta-sheet. Sequence comparison reveals a cluster of conserved amino acids that define a putative binding site with a buried zinc ion. Mutations of key residues in the putative active site result in loss of activity, enabling us to propose a catalytic mechanism. We also identified and synthesized muramyltripeptide, the minimal peptidoglycan fragment that can be used as a substrate by the enzyme. Molecular docking and digestion assays with muramyltripeptide derivatives allow us to identify key determinants of ligand binding. This results in a plausible model of interaction of this ligand not only for AmiE, but also for other PGN-hydrolases that share the same fold. As AmiE active-site mutations also show a severe growth defect, our findings provide an excellent platform for the design of specific inhibitors that target staphylococcal cell separation and can thereby prevent growth of this pathogen.

Show MeSH
Related in: MedlinePlus